Giuseppina Lacerra

β+45 G C: a novel silent β-thalassaemia mutation, the first in the Kozak sequence

A family from the Southeast of Italy was found to have a novel β‐globin mutant, β+45 G→C, with the features of a silent β‐thalassaemia mutation. It was asymptomatic in two heterozygotes, but its interaction with the severe thalassaemia mutation β‐IVS‐II‐654 C→T worsened the haematological and biosynthetic phenotype in two compound heterozygotes; moreover, another compound heterozygote, who was also heterozygote for the αααanti3·7, suffered from thalassaemia intermedia. The mutation was found associated in cis with the IVS‐II‐754 T→C substitution, which did not lead to abnormally spliced mRNA. Furthermore, the amount of β+45 mRNA was the same as the βA mRNA in the reticulocytes of the carriers. In vitro transcription/translation experiments demonstrated that the β+45 G→C decreased the efficiency of translation of the β‐globin chain by about 30%: this slight impairment was consistent with the observed clinical phenotype. The β+45 G→C is the first mutation found in the Kozak sequence (GACACCATGG) of the β‐globin gene and the first one at the position ‐6 upstream the ATG. The Kozak consensus sequence plays a major role in the initiation of translation process. The present finding supports the hypothesis that the G in position ‐6 is important in this process.

Hb Foggia or α117(GH5)Phe → Ser : a new α2 globin allele affecting the αHb-AHSP interaction

We report a novel α2-globin gene allele with the mutation cod 117 TTC>TCC or α117(GH5)Phe>Ser detected in three carriers with α-thalassemia phenotype. The mutated mRNA was present in the reticulocytes in the same amount as the normal one, but no chain or hemoglobin variant were detected. Most likely the amino acid substitution impairs the interaction of the α-chain variant with the AHSP and prevents its stabilizing effect, thus leading to the α-chain pool reduction.

Hb Bronte or α93(FG5)Val→Gly: A New Unstable Variant of the α2‐Globin Gene, Associated with a Mild α+‐Thalassemia Phenotype

We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides −181 to +894 (α1) and to +884 (α2) revealed a GTG→GGG substitution at codon 93 of the α2‐globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3–0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3–4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the α chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the α chains was much higher than that of the β‐globin chains, and the α/β biosynthetic ratio in the mother and proband was of the β‐thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H‐specific activity of the peak containing normal and variant α chains was not linear and was much higher than that of β chains; moreover, the α/β biosynthetic ratio varied during the 2 hours incubation.

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

SummaryWe report here a new human α-globin gene rearrangement carrying the two normal, α2 and α1, and two hybrid, α1/α2, globin genes in the order 5′-α2-α1/α2-α1/α2-α1-3′. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the β-thalassaemia IVS 1 nt 1 G→A mutation caused thalassaemia intermedia. We also report a case of an ααα-globin gene rearrangement in the twin of one of the αααα-globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an αααα-anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the α-globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.

β‐thalassaemia‐87 C→G: relationship of the Hb F modulation and polymorphisms in compound heterozygous patients

A clinical, haematological, biochemical and molecular study was carried out in 17 patients affected with thalassaemia intermedia, who were compound heterozygotes for the β‐thalassaemia mutation β‐87 C→G to determine the genetic basis of their clinical heterogeneity. The β‐87 was found associated with haplotype VIII (β‐87/VIII) or V (β‐87/V). The 10 patients with the β‐87/VIII showed milder clinical conditions, with significantly higher levels of haemoglobin (Hb) (9·8 ± 1·1 g/dl vs. 8·5 ± 1·3 g/dl) and fetal haemoglobin (Hb F) (6·2 ± 1·5 g/dl vs. 2·6 ± 1·5 g/dl; P = 0·0034) and higher synthesis of Gγ (Gγ/Totalγ 69·4 ± 2·6% vs. 42·8 ± 16·2%; P = 0·0042) than the seven patients with the β‐87/V. The β‐87/VIII showed a configuration of rare polymorphisms in the 5′ sub‐haplotype, which have been reported to exert an increasing effect on Hb F. They were ‘T’−158 Gγ‐globin gene, T‐A‐G in pre‐Gγ framework, (TG)11(CG)3 in the Gγ‐IVS2, (AT)9N12(AT)10 in LCR‐HS2; in contrast, the haplotype V had, respectively, ‘C’, T‐G‐A (TG)19(CG)2CACG in the Gγ‐IVS2, and (AT)10N12(AT)11. In all patients the β‐87 was associated with the (AT)9T5 motif 5′ β‐globin gene with increased affinity for the BP‐1 protein, and with the (TG)13 in the Aγ‐IVS2. The high increase of the Hb F, mostly of the Gγ‐type, strongly suggests the hypothesis that the ‘T’−158 Gγ plays a principal role and that the other polymorphisms could exert a cooperative role in the modulation of Hb F in patients with erythropoietic stress.

South-Italy β°-thalassemia: a novel deletion not removing the γ-globin silencing element and with 3′ breakpoint in a hsRTVL-H element, associated with β°-thalassemia and high levels of HbF

Hereditary persistence of fetal hemoglobin (HbF) in adulthood may be due to: mutations in the γ-globin genes promoter, mutations in the transcriptional factors involved in their regulation, polymorphic sequences or large deletions in the β-globin cluster.[1][1] Recently, Sankaran clarified the

Identification and molecular characterization of a novel 55-kb deletion recurrent in southern Italy: the Italian Gγ(Aγδβ)°-thalassemia†

To characterize the molecular basis of a β‐thalassemia defect in subjects with mild microcytosis associated with normal Hb A2 and increased levels of Hb F.

Hb City of Hope [β69(E13)Gly→Ser] in Italy : association of the gene with haplotype IX

Hb City of Hope [beta 69(E13)Gly----Ser] was detected by reversed phase high performance liquid chromatography in an asymptomatic carrier from Naples, Southern Italy. The amino acid substitution, identified by fast atom bombardment mass spectrometry, was due to a TGG----TGA substitution as assessed by DNA sequencing. Analysis of the chromosomal background indicates that the globin gene cluster containing the mutant gene has most probably been rearranged by a recombination event, since the mutation was associated with restriction fragment length polymorphism haplotype IX, instead of haplotype I, as previously reported.

HbA2-Partinico or δ(A2)Pro→Thr, a new genetic variation in the δ-globin gene in cis to the β+ thal IVS-I-110 G>A, and the heterogeneity of δ-globin alleles in double heterozygotes for β- and δ-globin gene defects

The study of the alleles of the δ-globin gene is relevant to the prevention of β-thalassemia homozygosis; in fact, the increase of the HbA2 is an invaluable hematological marker of the β-thalassemia heterozygosis and the double heterozygosis for alleles of δ- and β-globin genes can cause the decrease of the HbA2 up to normal or borderline values. We carried out the characterization of alleles of the δ- and β-globin genes, restriction fragment length polymorphism (RFLP) haplotype background, and hematologic phenotype in 23 double heterozygotes belonging to 18 unrelated families. A wide heterogeneity of the δ-globin alleles was detected; seven known alleles in trans to the β-globin gene defects were revealed in 17 out of 18 families, while a new allele in cis to a β-thalassemia allele was detected in one family. Moreover, the relative frequency of the δ-mutants was quite different from that found among heterozygotes. The new allele δ-cod 5 CCT>ACT, in cis to the allele β+ thal IVS-I-110 G>A, was found in five carriers of a Sicilian family. The new variant δ5(A2)Pro→Thr, named HbA2-Partinico upon the origin of the family, was detected with high-performance liquid chromatography; it overlapped the HbA2 peak which was partially split. The double in cis heterozygotes had increased percentage of normal and variant HbA2 of comparable size. The variant originated most likely from a new mutational event because it was associated with RFLP haplotype I, commonly found with the β+ thal IVS-I-110 G>A, even if crossing over or gene conversion cannot be excluded.

Identification and molecular characterization of a novel 163 kb deletion: The Italian (ϵγδβ)0-thalassemia

Objective and importance: To verify the presence of β-thalassemia in subjects showing hematologic phenotype of α-thalassemia, conduct normal molecular sequence analysis of the α-globin genes, and detect the absence of the most frequent α-thalassemia deletions. Clinical presentation: A patient from Apulia (Southern Italy) was referred to our institution for the occasional founding of hypochromic polyglobulia and microcytic red blood cells associated with normal levels of Hb A2 and Hb F and normal iron parameters. Intervention and technique: The patient has been investigated using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, restriction analysis, and gap-PCR. A novel deletion, the Italian (ϵγδβ)0-thalassemia, has been identified. The 5′ breakpoint was within a LINE element of 80 kb 3′ of the ε-globin gene, and the 3′ breakpoint was within a 160-bp palindrome of about 30 kb 5′ of the β-globin gene. The breakpoint region was characterized by the presence of a microhomology (5′-TCT-3′) and of an insertion of 43 bp owing to the duplication of the 160-bp palindrome. Comparison of the Hb and Hb A2 values of (ϵγδβ)0-thalassemia from the literature with those of (molecularly known) thalassemia carriers indicated a higher level of Hb A2 with respect to α-thalassemia and a lower level of Hb with respect to β0-thalassemia carriers. Conclusion: In this study, we report the first (ϵγδβ)0-thalassemia case identified in Italy. To avoid misdiagnosis of β-thalassemia, we suggest verifying the presence of large deletions of the β-globin gene cluster in subjects showing a higher border line level of Hb A2 and a lower level of Hb.